Evaporative light scattering detection is a method of detecting samples that have been previously separated in various chromatography methods such as, for example, High Performance Liquid Chromatography (HPLC), Gel-Permeation Chromatography (GPC), High Performance Centrifugal Partition Chromatography (PCPC), Field Flow Fractionation (FFF), and Supercritical Fluid Chromatography (SFC). Evaporative light scattering detection is preferably used when the sample components (e.g., components to be detected) have lower volatility than the mobile phase. A wide variety of sample types can be detected in evaporative light scattering detection. Such sample types include, for example, lipids, triglycerides, surfactants, polymers, underivatized fatty and amino acids, carbohydrates and pharmaceuticals.
Generally, evaporative light scattering detection involves four main steps: 1) nebulization of the chromatography effluent, (which consists of the mobile phase and the sample), into an aerosol of particles, 2) evaporation of the mobile phase, 3) light scattering by the sample particles, and 4) detection of the scattered light. There are two principal types of devices used in evaporative light scattering detection known in the art. In the first type (the “single flow” design), the nebulized chromatography effluent is immediately introduced into a heated drift tube where the mobile phase is evaporated. The sample particles are then flowed from the heated drift tube to an optical cell where light scattering and detection occurs. One such example of this type of device (the Alltech Model 500 ELSD) is sold by the assignee of this application, ALLTECH ASSOCIATES, INC. Details concerning the design and operating parameters for such a device are disclosed in the Operating Manual for the Alltech Model 500 ELSD, which is incorporated herein by reference.
In the second type of device, (the “split-flow” design), the nebulized chromatography effluent is first flowed through a nebulization chamber before entering the heated drift tube. In the nebulization chamber, the nebulized chromatography effluent is split, namely, the larger droplets are eliminated by condensation/impaction on the walls of the nebulization chamber. This condensate is drained to waste. Only the smaller nebulized droplets are subsequently flowed to the drift tube where the mobile phase (which is now free of the larger droplets) is more easily evaporated. Thereafter, the sample particles are flowed to the optical cell for light scattering and detection. Devices of this design type are available from, for example, SEDERE or EUROPSEP INSTRUMENTS.
The above-described design types have particular advantages depending on the mobile phase and the sample type. The single flow design is preferred for use in applications involving relatively non-volatile sample types and volatile organic mobile phases. Because all of the sample enters the optical cell in this design, response and sensitivity is maximized.
However, the split-flow design is preferably used with highly aqueous mobile phases and semi-volatile sample types. Highly aqueous mobile phases generally require higher evaporation temperatures. If the sample is volatile at these higher evaporation temperatures, sample loss is incurred during the evaporation step resulting in poorer sensitivity. By using the split-flow design, the evaporation temperature is reduced. This is accomplished by removing the larger mobile phase droplets in the nebulized chromatography effluent before the evaporation step. By removing the larger droplets, a smaller and more uniform particle size distribution is achieved in the mobile phase, which leads to lower evaporation temperatures. The lower evaporation temperatures, in turn, lead to less sample loss during the evaporation step. However, for non-volatile sample types and organic mobile phases, the split-flow design is generally less preferred because some of the non-volatile sample may be lost during the splitting of the chromatography effluent.
Another problem with devices of the split-flow design is that the split ratio of the sample (i.e., the amount that goes to waste versus the amount that is ultimately detected) is affected by, among other things, the laboratory temperature. In other words, fluctuations in laboratory temperatures lead to fluctuations in droplet size in the nebulized chromatography effluent. Thus, as ambient and/or laboratory temperatures fluctuate, the split ratio and corresponding reproducibility of sample detection may vary from run to run.
As is evident from the above-discussion, depending on the mobile phase and the sample type being detected, one evaporative light scattering detection design and method is advantageous over the other. However, laboratories often work with both aqueous and organic mobile phases and various sample types with different volatilities. Ideally, laboratories would have available both design types for evaporative light scattering detection. However, in order to have this benefit, the laboratory would need to purchase two separate devices, which can be expensive. It would be advantageous and constitute an improvement in the art if an evaporative light scattering detection device and system were developed which could be quickly and inexpensively converted between the single flow and split flow designs. Applicants have developed such a device and system. Moreover, with respect to the split-flow design, Applicants invention addresses the problem of the variation in split ratio caused by fluctuating laboratory temperatures.